Multicellular organisms have developed two general systems of immunity to infectious agents. The two systems are innate or natural immunity (usually referred to as “innate immunity”) and adaptive (acquired) or specific immunity. The major difference between the two systems is the mechanism by which they recognize infectious agents. Recent studies have demonstrated that the innate immune system plays a crucial role in the control of initiation of the adaptive immune response and in the induction of appropriate cell effector responses (Fearon et al. Science 1996; 272:50-53 and Medzhitov et al. Cell 1997; 91:295-298).
The innate immune system uses a set of germline-encoded receptors for the recognition of conserved molecular patterns present in microorganisms. These molecular patterns occur in certain constituents of microorganisms including: lipopolysaccharides, peptidoglycans, lipoteichoic acids, phosphatidyl cholines, bacterial proteins (e.g., flagellin), including lipoproteins, bacterial DNAs, viral single and double-stranded RNAs, unmethylated CpG-DNAs, mannans, and a variety of other bacterial and fungal cell wall components. Such molecular patterns can also occur in other molecules such as plant alkaloids. These targets of innate immune recognition are called Pathogen Associated Molecular Patterns (PAMPs) since they are produced by microorganisms and not by the infected host organism (Janeway et al. Cold Spring Harb. Symp. Quant. Biol. 1989; 54:1-13 and Medzhitov et al. Curr. Opin Immunol. 1997; 94:4-9). PAMPs are discrete molecular structures that are shared by a large group of microorganisms. They are conserved products of microbial metabolism, which are not subject to antigenic variability (Medzhitov et al. Cur Op Immun 1997; 9:4).
The receptors of the innate immune system that recognize PAMPs are called Pattern Recognition Receptors (PRRs) (Janeway et al. Cold Spring Harb. Symp. Quant. Biol. 1989; 54:1-13 and Medzhitov et al. Curr. Opin. Immunol. 1997; 94:4-9). These receptors vary in structure and belong to several different protein families. Some of these receptors recognize PAMPs directly (e.g., TLR3, collectins), while others (e.g., complement receptors) recognize the products generated by PAMP recognition.
Cellular PRRs are expressed on effector cells of the innate immune system, including cells that function as professional antigen-presenting cells (APC) in adaptive immunity. Such effector cells include, but are not limited to, macrophages, dendritic cells, B lymphocytes, and surface epithelia. This expression profile allows PRRs to directly induce innate effector mechanisms, and also to alert the host organism to the presence of infectious agents by inducing the expression of a set of endogenous signals, such as inflammatory cytokines and chemokines. This latter function allows efficient mobilization of effector forces to combat the invaders. Examples of PRRs include Nod-like receptors (NLRs) and Toll-like receptors (TLRs).
NLRs are cytoplasmic proteins that may have a variety of functions in regulation of inflammatory and apoptotic responses. NLRs are composed of conserved “modules” including a central nucleotide-binding oligomerization domain and a series of tandem leucine-rich repeats. NLRs are encoded by genes from a large gene family present in many different animal species; there are more than 20 NLR genes in humans. Many are thought to serve as PRRs which sense microbial products in the cytoplasm of cells, although some members have different functions. The ligands are currently known for the NLRs, NLRC1 (NOD1) and NLRC2 (NOD2). NLRC1 recognizes a molecule called Meso-diaminopimelic acid (meso-DAP), which is a peptidoglycan constituent of only Gram negative bacteria. NLRC2 proteins recognize intracellular MDP (muramyl dipeptide), which is a peptidoglycan constituent of both Gram positive and Gram negative bacteria. These proteins transduce signals in the pathway of NF-κB and MAP kinases. To do this, they interact with the serine-threonine kinase called RIPK2 via an N-terminal CARD domains and interact with microbial molecules by means of a C-terminal leucine-rich repeat (LRR) region [Strober et al., Signalling pathways and molecular interactions of NOD 1 and NOD2. Nat Rev Immunol. 2006, Volume 6(1):9-20.] NLRC4 (IPAF) has also been shown to activate caspase-1 in response to bacteria. Further, anthrax toxin activates NLRP1 (previously called NALP1), and Staphylococcus aureus toxins such as alpha-hemolysin (GenBank Accession No. AAA26598) (SEQ ID NO: 1) activate NLRP3. Other NLRs such as NAIP have also been shown to activate caspase-1 in response to Salmonella and Legionella. [SEE, Inohara et al., NOD-LRR proteins: role in host-microbial interactions and inflammatory disease. Annu Rev Biochem. 2005, Volume 74:355-83; Strober et al., Signalling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol. 2006, Volume 6(1):9-20; Chen G, Shaw M H, Kim Y G, Nuñez G. Annu Rev Pathol. 2009, 4:365-98; Martinon F, Mayor A, Tschopp J. Annu Rev Immunol. 2009; 27:229-65].
The best characterized class of cellular PRRs are members of the family of Toll-like receptors (TLRs), so called because they are homologous to the Drosophila Toll protein which is involved both in dorsoventral patterning in Drosophila embryos and in the immune response in adult flies (Lemaitre et al. Cell 1996; 86:973-83). At least 12 mammalian TLRs, TLRs 1 through 11 and TLR13, have been identified to date (see, for example, Medzhitov et al. Nature 1997; 388:394-397; Rock et al. Proc Natl Acad Sci USA 1998; 95:588-593; Takeuchi et al. Gene 1999; 231:59-65; and Chuang and Ulevitch. Biochim Biophys Acta. 2001; 1518:157-61). Activation of signal transduction pathways by TLRs leads to the induction of various genes including inflammatory cytokines, chemokines, major histocompatability complex, and co-stimulatory molecules (e.g., B7). For example, activation of TLR4 can induce the secretion of tumor necrosis factor (TNF) and of the interleukins IL-1 and IL-6 as part of an antibacterial response, and can induce the secretion of the interferons INFα and INFβ as part of an anti viral response.
TLR signaling consists of at least two distinct pathways: a MyD88-dependent pathway that leads to the production of inflammatory cytokines, and a MyD88-independent pathway associated with the stimulation of IFN-β and the maturation of dendritic cells. The MyD88-dependent pathway is common to all TLRs, except TLR3 [Adachi O. et al., 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity. 9(1):143-50.]. Upon activation by microbial antigens, TLRs induce the recruitment of MyD88 via its TIR domain which in turn recruits IRAK1 and IRAK4 and leads to complex downstream signaling cascades leading to the phosphorylation of IκB and the subsequent nuclear localization of NF-κB. Activation of NF-κB triggers the production of pro-inflammatory cytokines such as TNF-α, IL-1 and IL-12.
In mammalian organisms, TLRs have been shown to recognize PAMPs such as the bacterial products LPS (Schwandner et al. J. Biol. Chem. 1999; 274:17406-9 and Hoshino et al. J. Immunol. 1999; 162:3749-3752), lipoteichoic acid (Schwandner et al. J. Biol. Chem. 1999; 274:17406-9), peptidoglycan (Yoshimura et al. J. Immunol. 1999; 163:1-5), lipoprotein (Aliprantis et al. Science 1999; 285:736-9), CpG-DNA (Hemmi et al. Nature 2000; 408:740-745), and flagellin (Hayashi et al. Nature 2001; 410:1099-1103), as well as the viral product double stranded RNA (Alexopoulou et al. Nature 2001; 413:732-738) and the yeast product zymosan (Underhill. J Endotoxin Res. 2003; 9:176-80). For example, TLR2 is essential for the recognition of a variety of PAMPs, including bacterial lipoproteins, peptidoglycan, and lipoteichoic acids. TLR3 is implicated in virus-derived double-stranded RNA. TLR4 is predominantly activated by lipopolysaccharide. TLR9 is required for response to unmethylated CpG DNA. Recently, TLR7 and TLR8 have been shown to recognize single stranded RNA molecules (Hornung V. et al. Handb Exp Pharmacol. 2008; (183):71-86), and small synthetic antiviral molecules (Jurk M. et al. Nat Immunol 2002; 3:499). TLR11 detects profilin-like protein (PLP). Furthermore, TLR5 detects bacterial flagellin.
Flagellin is a protein expressed by a variety of flagellated bacteria (Salmonella typhimurium for example) as well as non-flagellated bacteria (such as Escherichia coli). Sensing of flagellin by cells of the innate immune system (dendritic cells, macrophages, etc) is mediated by the Toll-like receptor 5 (TLR5) as well as by Nod-like receptors (NLRs) Ipaf and Naip5 (Franchi et al (2006) Nat Immunol 7(6):576-582; Miao et al (2006) Nat Immunol 7(6):569-575; and Ren et al (2006) PLoS Pathog 2(3):e18). Various reports have described the role of TLRs and NLRs in the activation of innate immune response and adaptive immune response. Thus, it has been suggested that flagellin, like other TLR ligands, could be a relevant adjuvant in immunotherapies.
Bacillus anthracis is the bacterium that causes anthrax. The bacterium secretes a toxin called anthrax lethal toxin, which is the major cause of pathogenesis, and is composed of a protective antigen and a lethal factor (Stephen, J. Anthrax toxin. 1981. Pharmacol. Ther. 12, 501-513). It was recently shown that lethal factor component of anthrax toxin enters the cytosol of macrophages and other cell types, and is recognized by the NLR protein Nalp1 or NLRP1 and mediates cell death (Boyden et al. 2006 Nat Genet. 38:240-244).
Staphylococcus aureus is a Gram positive bacterium responsible for a wide variety of superficial as well as serious life-threatening infections (Lowy et al. 1998. N. Engl. J. Med. 339: 520-525). S. aureus secretes many toxins among which α-hemolysin has been implicated in the pathogenesis of S. aureus necrotizing pneumonia and various other symptoms in animal models. α-Hemolysin is secreted as a 33-kDa monomer and oligomerizes, forming heptameric transmembrane pores (Song et al. 1996 Science 274: 1859-1866). It was recently shown that α-hemolysin is recognized by the NLR-protein NLRP3, and initiates cell death (Craven et al. 2009 PLoS One 4:e7446; Munoz-Planillo et al. 2009 183:3942-3948).
TLR ligands have been exploited as adjuvant in numerous therapy regimens [Koski, G. K. et al., Reengineering dendritic cell-based anti-cancer vaccines. Immunol Rev 222, 256 (2008)]. For example, local administration of live bacilli Calmette-Guerin (BCG), which stimulate TLR2 and TLR4, has been proven to be beneficial in the treatment of tumors such as bladder cancer [Herr, H. W. et al., Intravesical bacillus Calmette-Guerin therapy prevents tumor progression and death from superficial bladder cancer: ten-year follow-up of a prospective randomized trial. J Clin Oncol 13 (6), 1404 (1995)]. Imiquimod, an agonist for TLR7, is approved for the treatment of basal cell carcinoma and precursor lesion of cutaneous squamous cell carcinoma [Herr, H. W. et al., Intravesical bacillus Calmette-Guerin therapy prevents tumor progression and death from superficial bladder cancer: ten-year follow-up of a prospective randomized trial. J Clin Oncol 13 (6), 1404 (1995)]. Similarly, the TLR9 ligand CpG has also been used in different mono-therapies, combination therapies and Phase I/II trials [Dougan, M. and Dranoff, G., Immune Therapy for Cancer. Annu Rev Immunol (2008)].
In peptides-based vaccines, the use of TLR ligands in conjunction with long peptides containing helper and cytotoxic T lymphocytes (CTL) epitopes, has shown to be efficient at promoting helper CD4+ T cells [Melief, C. J. et al., Effective therapeutic anticancer vaccines based on precision guiding of cytolytic T lymphocytes. Immunol Rev 188, 177 (2002); Jackson, D. C. et al., A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic T cell responses. Proc Natl Acad Sci USA 101 (43), 15440 (2004)]. Similarly to TLR ligands, many adjuvants used in human vaccine also include ligands for NLRs and can activate DCs [Martinon, F., Mayor, A., and Tschopp, J., The inflammasomes: guardians of the body. Annu Rev Immunol 27, 229 (2009)]. Further, recently it has been discovered that TLR ligands enhance presentation of phagocytosed antigens within major histocompatibility class II MHC molecules [Blander, J. M. and Medzhitov, R., Nature (2006), Vol. 440, pp. 808].
Recent evidence demonstrates that fusing a polypeptide ligand specific for a Toll-like receptor (TLR) to an antigen of interest generates a vaccine that is more potent and selective than the antigen alone. It has been previously shown that immunization with recombinant TLR-ligand:antigen fusion proteins: a) induces antigen-specific T-cell and B-cell responses comparable to those induced by the use of conventional adjuvant, b) results in significantly reduced non-specific inflammation; and c) results in CD8+ T-cell-mediated protection that is specific for the fused antigen epitopes (See, for example US published patent applications 2002/0061312 and 2003/0232055 to Medzhitov, and US published patent application 2003/0175287 to Medzhitov and Kopp). For example, mice immunized with a fusion protein consisting of the polypeptide PAMP BLP linked to Leishmania major antigens mounted a Type 1 immune response characterized by antigen-induced production of γ-interferon and antigen-specific IgG2a (Cote-Sierra et al. Infect Immun 2002; 70:240-248). The response was protective, as demonstrated in experiments in which immunized mice developed smaller lesions than control mice did following challenge with live L. major. Furthermore, flagellin fusion to well defined antigens promotes protective immunity in mice [Huleatt, J. W. et al., in Vaccine (2007), Vol. 25, pp. 763; Huleatt, J. W. et al., in Vaccine (2008), Vol. 26, pp. 201.] and activates human DCs [Arimilli, S. et al., Engineered Expression of the TLR5Ligand Flagellin Enhances Paramyxovirus Activation of Human Dendritic Cell Function. J Virol (2008)].
While the above fusion proteins provided a lot of promise based on their in vitro data, thus far it has proven difficult to achieve long-lasting, effective immunity, including generation of both CD4+ and CD8+ T cell responses, to the desired antigen in clinical trials using such fusion proteins. CD8+ T cells (such as cytotoxic T lymphocytes (CTLs)) directly kill tumor cells and are important for tumor rejection. CD4 T helper (Th) cell responses can also contribute to anti-tumor activity through direct killing of tumors, by supporting both the activation and long-term maintenance of CD8+ T cells, and through the production of cytokines. Th cells can also support the humoral immune response mediated by B cells [Koski et al. (2008) supra].
Immunotherapy, if successful, would be particularly appealing for use as a cancer treatment, for which new and better treatments are desperately needed. The last decade has witnessed steady reductions in the death rates for many types of cancer. These reductions are largely due to improvements in early detection, advanced surgical techniques, refinements in the administration of radiation therapies, and the discovery of new, molecular-targeted chemotherapeutic agents. However, countless instances occur either where tumors are not amenable to any existing therapy or they respond initially only to recur in forms resistant to front-line therapies, leaving limited treatment options. Moreover, immunotherapies would be suitable to prevent relapse.
The development of novel treatment modalities will greatly benefit cancer patients. One such modality is immunotherapy, which posits that the immune system can be enlisted in the fight against cancer. There has existed for some time compelling evidence that cellular and molecular agents of the immune system are capable of attacking tumors, and experimental immunotherapeutic interventions have sought to take advantage of each of them. Although most immunotherapy trials have yielded somewhat disappointing results, there are some examples of success, such as recent T-cell adoptive therapy trials. These treatments have proven that immunotherapy can induce pronounced tumor regressions that are associated with prolonged survival for advanced melanoma [Koski et al. (2008) Immunological Reviews 222:256-276]. At least for relatively advanced melanoma, such outcomes are currently superior to any other therapeutic modality available. However, this type of therapy involves the cultivation of huge numbers of patient lymphocytes, which requires uncommon technical expertise and specialized facilities. Therefore, less labor intensive forms of immunotherapy, such as vaccine modalities, are desirable for more widespread implementation.
Unfortunately, vaccine strategies have underperformed these more labor-intensive adoptive immunotherapy approaches. Breakthroughs in the understanding of tumor immunology are needed to advance vaccine-based immunotherapy to this next level. One substantial hope for the development of cancer vaccines came with the development of methods to culture human and mouse dendritic cells (DCs) [Koski et al. (2008) supra]. Because DCs were considered the most efficient known cells for the presentation of antigen to T cells, it was therefore supposed (based on some early work with murine models) that it might be relatively easy to pulse tumor antigens onto DCs and use these cells to successfully vaccinate against tumors [Zitvogel L, et al. Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulatiori, and T helper cell 1-associated cytokines. J Exp Med 1996; 183:87-97.]. The primary source for human DC precursors was the blood and bone marrow, but the first methods produced only immature DCs. Later, ways were found to mature these cells, which usually involved a second step culture with additional cytokines [Zhou L F, Tedder T F. CD141 blood monocytes can differentiate into functionally mature CD831 dendritic cells. Proc Natl Acad Sci USA 1996; 93:2588-2592.]. Both immature and mature cells have been tested in clinical trials to treat various malignancies. Whereas occasional clinically relevant responses were observed, the overall results have been disappointing [Koski et al. (2008) supra].
There is therefore a need to develop improved compositions and methods for cancer immunotherapy. The present invention provides such methods.